Various epigenetic marks at the HIV-1 5'LTR suppress proviral expression and promote latency. Cellular antisense transcripts known as long noncoding RNAs (lncRNAs) recruit the polycomb repressor complex 2 (PRC2) to gene promoters, which catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3), thus promoting nucleosome assembly and suppressing gene expression. We found that an HIV-1 antisense transcript expressed from the 3'LTR and encoding the antisense protein ASP promotes proviral latency. Expression of ASP RNA reduced HIV-1 replication in Jurkat cells. Moreover, ASP RNA expression promoted the establishment and maintenance of HIV-1 latency in Jurkat E4 cells. We show that this transcript interacts with and recruits PRC2 to the HIV-1 5'LTR, increasing accumulation of the suppressive epigenetic mark H3K27me3, while reducing RNA Polymerase II and thus proviral transcription. Altogether, our results suggest that the HIV-1 ASP transcript promotes epigenetic silencing of the HIV-1 5'LTR and proviral latency through the PRC2 pathway.

Circulating plasmacytoid dendritic cells (pDC) decline during HIV-1 infection, but at the same time they express markedly higher levels of interferon alpha (IFNα), which is associated with HIV-1 disease progression. Here we show an accumulation of pDC in lymph nodes (LN) of treatment-naïve HIV-1 patients. This phenomenon was associated with elevated expression of the LN homing marker, CCR7, on pDC in peripheral blood of HIV-1 patients, which conferred increased migratory capacity in response to CCR7 ligands in ex vivo functional assays. LN-homed pDC of HIV-1 patients presented higher CD40 and lower BDCA2 levels, but unchanged CD83 and CD86 expression. In addition, these cells expressed markedly higher amounts of IFNα compared to uninfected individuals, and were undergoing faster rates of cell death. These results demonstrate for the first time that in asymptomatic, untreated HIV-1 patients circulating pDC up-regulate CCR7 expression, accumulate in lymph nodes, and express high amounts of IFNα before undergoing cell death. Since IFNα inhibits cell proliferation and modulates immune responses, chronically high levels of this cytokine in LN of HIV-1 patients may impair differentiation and immune function of bystander CD4⁺ T cells, thus playing into the mechanisms of AIDS immunopathogenesis.

Resting memory CD4⁺T cells are the largest reservoir of persistent infection in HIV-1-positive subjects. They harbor dormant, stably integrated virus despite suppressive antiretroviral therapy, posing an obstacle to a cure. Surface markers that identify latently infected cells remain unknown. Microarray analyses comparing resting latently infected and uninfected CD4⁺T cells generatedin vitroshowed profound differences in the expression of gene programs related to transcriptional and posttranscriptional regulation, cell proliferation, survival, cycle progression, and basic metabolism, suggesting that multiple biochemical and metabolic blocks contribute to preventing viral production in latently infected cells. We identified 33 transcripts encoding cell surface markers that are differentially expressed between latently infected and uninfected cells. Quantitative reverse transcriptase PCR (RT-QPCR) and flow cytometry analyses confirmed that the surface marker CD2 was expressed at higher levels on latently infected cells. To validate this resultin vivo, we sorted resting memory CD4⁺T cells expressing high and low surface levels of CD2 from six HIV-1-infected subjects successfully treated with antiretroviral drugs for at least 3 years. Resting memory CD4⁺CD2^highT cells from all subjects harbored higher HIV-1 DNA copy numbers than all other CD4⁺T cell subsets. Moreover, afterex vivoviral reactivation, robust viral RNA production was detected only from resting memory CD4⁺CD2^highT cells but not from other cell subsets. Altogether, these results show that a high CD2 expression level is a hallmark of latently infected resting memory CD4⁺T cellsin vivo.

Background: The role of plasmacytoid dendritic cells (pDC) and interferon alpha (IFNα) in HIV-1 infection is still unclear. On one hand, HIV-1 disease is associated with a progressive decline of pDC, which displays reduced ability to produce IFNα after in vitro challenge. On the other hand, high IFNα serum levels in HIV-1-infected individuals have been proposed to promote immune hyper-activation and disease progression. Methods: We sought to determine whether disappearance of pDC in HIV-1 disease is due to homing in lymphoid tissues. We also studied IFNα and myxovirus resistance protein A (MxA) expression in unstimulated pDC and correlated these results with selected clinical and laboratory parameters. Results: We found that pDC decline markedly in peripheral blood of patients progressing to disease but at the same time express much higher levels of IFNα and MxA compared to control individuals. On the other hand, we observed steady pDC counts in lymph nodes of HIV-1 patients. The frequency of circulating pDC correlated directly with CD4 cell counts and inversely with viral load. However, we found no correlation between IFNα and MxA expression levels, CD4 counts, and viral load. Conclusions: Circulating pDC decline sharply in the course of HIV-1 disease, but express high levels of IFNα, which may represent a hallmark of systemic immune dysfunction.

The negative strand of HIV-1 encodes a highly hydrophobic antisense protein (ASP) with no known homologs. The presence of humoral and cellular immune responses against ASP in HIV-1 patients indicate that ASP is expressed in vivo, but its role in HIV-1 replication remains unknown. We investigated ASP expression in multiple chronically infected myeloid and lymphoid cell lines using an anti-ASP monoclonal antibody (324.6) in combination with flow cytometry and microscopy approaches. At baseline and in the absence of stimuli, ASP shows a polarized sub-nuclear distribution, preferentially in areas with low content of suppressive epigenetic marks. However, following treatment with phorbol 12-myristate 13-acetate (PMA), ASP translocates to the cytoplasm, and is detectable on the cell surface even in the absence of membrane permeabilization, indicating that 324.6 recognizes an ASP epitope that is exposed extracellularly. Further, surface staining with 324.6 and anti-gp120 antibodies showed that ASP and gp120 co-localize, suggesting that ASP might become incorporated in the membrane of budding virions. Indeed, fluorescence correlation spectroscopy studies showed binding of 324.6 to cell-free HIV-1 particles. Moreover, 324.6 was able to capture and retain HIV-1 virions with efficiency similar to anti-gp120 VRC01. Our studies indicate that ASP is an integral protein of the plasma membrane of chronically infected cells stimulated with PMA, and upon viral budding ASP becomes a structural protein of the HIV-1 envelope. These results may provide leads to investigate the possible role of ASP in the virus replication cycle, and suggest that ASP may represent a new therapeutic or vaccine target. IMPORTANCE The HIV-1 genome contains a gene expressed in the opposite or antisense direction of all other genes. The protein product of this antisense gene, called ASP, is poorly characterized and its role in viral replication remains still unknown. We provide evidence that the antisense protein, ASP of HIV-1 is found within the cell nucleus of unstimulated cells. In addition, we show that after PMA treatment ASP exits the nucleus and localizes on the cell membrane. Moreover, we demonstrated that ASP is present on the surface of viral particles. Altogether, our studies identify ASP as a new structural component of the HIV-1 virus, and show that ASP is an accessory protein that promotes viral replication. The presence of ASP on the surface of both infected cells and viral particles might be exploited therapeutically.

Humans tailor virus-specific immune responses through modulated expression of 12 different interferon (IFN)-α subtypes. However, exacerbated expression of certain IFN-α subtypes causes immunopathology in the context of autoimmune conditions and chronic viral infections. We showed that progression to AIDS is associated with elevated expression of IFN-α in unstimulated peripheral blood mononuclear cells. Here, we sought to determine whether distinct IFN-α subtypes are involved in this phenomenon. We used quantitative RT-PCR to assess expression levels of 12 IFN-α subtypes in peripheral blood mononuclear cells from normal donors and HIV-1 patients at CDC stage A and stage C of the disease. Three patterns of IFN-α subtype expression emerged. First, IFN-α2 and IFN-α6 mRNA levels were elevated in both patient groups. Second, IFN-α1/13, IFN-α8, IFN-α14, IFN-α16, IFN-α17, and IFN-α21 were upregulated in stage C but not stage A patients. Third, expression levels of IFN-α4, IFN-α5, IFN-α7, and IFN-α10 did not change among the three groups of volunteers. Among all other subtypes, IFN-α2 was preferentially upregulated, showing >60-fold higher levels in stage A and >400-fold in stage C patients compared with controls, which correlated with declining CD4 counts. Our results demonstrate that distinct IFN-α subtypes are sequentially activated during HIV-1 infection, which may be predictive of disease progression.